Chiral separation of enantiomers by high-speed countercurrent chromatography

ABSTRACT

Preparative-scale separations of chiral compounds were achieved by high-speed countercurrent chromatography (HSCCC) using a multilayer coil planet centrifuge equipped with a 325 mL capacity column. The separations were performed by two different procedures both utilizing a set of N-(3,5-introbenzoyl)-D,L-amino acids as test samples with N-dodecanoyl-L-proline-3,5-dimethylanilide as a chiral selector (Cs). The HSCCC separations were carried out with a two-phase solvent system composed of hexane/ethyl acetate/methanol/water where the chiral selector was added to the organic stationary phase. A second procedure using pH-zone-refining CCC yielded characteristic fused rectangular peaks in which the two isomers were resolved with less than 5% of overlap.

REFERENCE TO RELATED APPLICATIONS

This application is a division of Ser. No. 08/357,845, filed Dec. 16,1994, now U.S. Pat. No. 6,337,021.

This invention lies in the field of liquid-liquid partitionchromatography, and in particular in the chiral separation ofenantiomers using chromatographic techniques.

BACKGROUND OF THE INVENTION

Countercurrent chromatography (CCC) is a form of liquid-liquid partitionchromatography which relies on the continuous contact between twoimmiscible solvents, one of which is mobile relative to the other, in aflow-through tubular column, free of any solid support matrix. Theretention time of a solute in the phase contact region of the system isdetermined by the volume ratio of the solvents, the partitioncoefficient of the solute between the solvents, and the degree ofcontact between the solvents. Like other forms of liquid-liquidpartition chromatography, one of the solvents serves as a carrier,drawing the solutes from the other solvent and carrying the solutes outof the column in the order of elution. This carrier solvent is thusreferred to as the mobile phase, while the other solvent is referred toas the stationary phase, even though it is not strictly stationary inmany applications of the method. Solvent mixing, retention of thestationary phase in the column, and solute partitioning all take placein the column by the aid of a suitable acceleration field established bygravity, centrifugal force or both, and the configuration of the column.

Most equipment used for CCC separations involves a coil of columntubing, a portion of which is filled with the stationary phase while themobile phase is passed through it. By varying the length and diameter ofthe tubing, CCC has been used for both analytical and preparativeseparations.

The flow rate of the mobile phase may be varied by varying the fieldimposed on the column. Units which operate in the presence of agravitational field only are restricted to slow flow rates, with theresulting separations typically requiring 1 to 3 days, to avoiddisplacing the stationary phase. A unit which operates in the presenceof a centrifugal acceleration field of 40 g or more allows faster flowrates and permits separation times of only a few hours.

Separations by CCC may be performed using any immiscible pair ofsolvents, provided that the solvents differ in density to at least aslight degree. Both normal-phase and reverse-phase separations may beperformed, with the more polar solvent as the stationary phase fornormal-phase separations, and the less polar solvent as the stationaryphase for reverse-phase separations.

The operational aspects of CCC are similar to the more conventionalliquid-liquid chromatography (LLC). Typically, after the immisciblesolvent phases are equilibrated relative to one another, the column isfilled with the stationary phase. The sample is then injected into thecolumn and elution with the mobile phase is begun. The centrifuge isthen stared and the eluting fractions are collected. Initially, thefractions are composed of the stationary phase which is displaced fromthe column. However, once hydrodynamic equilibrium between the phases isachieved, only small portions of the stationary phase will co-elute withthe mobile phase. The effluent is continuously monitored with a uvdetector and fractionated into test tubes using a fraction collector.The collected fractions are monitored by any of a variety of meansincluding spectroscopic methods and thin-layer chromatography.

Countercurrent chromatographic theory, as well as apparatus forperforming the method, are described by Ito, Y., in “Principle andInstrumentation of Countercurrent Chromatography,” in CountercurrentChromatography: Theory and Practice Mandava, N. B., and Ito, Y., eds.,pp. 79-442 (Marcel Dekker, New York, 1988) and by Conway, W. D., inCountercurrent Chromatography: Apparatus, Theory and Applications (VCH,New York, 1990). Most countercurrent chromatographs use a column whichis formed into a helical coil. This coil is in turn mounted onto acolumn holder in various configurations relative to the means forrotating it and relative to the acceleration field that acts on it.

Each column and each type of rotation produce different types of mixingbetween the solvent phases and are particularly suited for specificseparations. However, certain disadvantages to CCC exist.

One disadvantage associated with CCC is the increased peak widthassociated with increased retention time of the solute. This increasedpeak width makes detection of the solute more difficult, and requires alarger volume of eluate to be collected and processed in order to obtaina maximum yield of solute. This disadvantage is particularly acute whenpreparative separations are desired. Nevertheless, increased retentiontime is desirable in order to avoid coeluting impurities with thesolute. Commonly-owned, copending U.S. patent application Ser. No.07/946,613, filed Sep. 18, 1992, discloses a method for obtaining sharpelution peaks in analytical or semi-preparative CCC without decreasingthe retention time of the solute, by adding a peak sharpening agent toeither the stationary phase or the sample mixture. When acidic compoundsare to be separated, the peak sharpening agent is an acid. When basicsolutes are to be separated, the peak sharpening agent is a base.

More recently, an unusually efficient separation of mixtures of acids orbases has been described using a unique modification of the techniquesof countercurrent chromatography. See, Ito, et al. U.S. Pat. No.5,332,504, the disclosure of which is incorporated herein by reference.According to this modification, the two immiscible liquid solutionswhich are to serve as the stationary and mobile phases, respectively,are modified prior to the performance of the separation by rendering oneof the phases acidic and the other basic. Separation of a mixture ofacids is then performed in a system in which the acidified solutionserves as the stationary phase and the basified solution as the mobilephase. Conversely, separation of a mixture of bases is performed in asystem in which the basified solution serves as the stationary phase andthe acidified solution as the mobile phase. Individual acid or basicsolutes separated by this method elute in contiguous, well-resolved,rectangularly shaped peaks, the solutes eluting in order of their pK_(a)values and hydrophobicity and the fractions within any single peak beingof substantially constant concentration. The combined fractions withineach peak differ in pH, successively increasing in the case of a basicmobile phase and successively decreasing in the case of an acidic mobilephase. For this reason, the technique has been referred to as“pH-zone-refining countercurrent chromatography.”

A recent modification of pH-zone-refining countercurrent chromatographyis carried out in a manner analogous to displacement chromatography.See, commonly-owned, copending U.S. patent application Ser. No.08/263,924, filed Jun. 21, 1994 and incorporated herein by reference.This method uses a retainer base (acid) in the stationary phase toretain analytes in the column and a displacer acid (base) to elute theanalytes in the decreasing (or increasing) order of pK_(a) andhydrophobicity. The elution produces a train of highly concentratedrectangular solute peaks with minimum overlap. To use pH-zone-refiningCCC in a displacement mode, the mobile and stationary phases areswitched. Thus, the original eluent becomes a retainer to retainanalytes in the stationary phase, and the original retainer acid becomesa displacer to displace the analytes from the stationary phase to themobile phase at the back of the solute bands.

Displacement countercurrent chromatography and pH-zone-refiningcountercurrent chromatography (in the normal mode) both entail certainadvantages over previously known counter-current chromatographytechniques. First, the method permits one to load the sample as asuspension into the separation column. Thus, mixtures of compounds thatare only partially soluble in the solvent system can be separatedefficiently. In addition, the lack or small degree of elution peakoverlap permits one to separate mixtures of greater volume than beforein any given column without loss of resolution. For example, columnswhich are otherwise recommended for separations of mixtures of a certainmaximum size can be used for separating mixtures up to ten times thatsize or greater. Likewise, mixtures containing higher concentrations ofthe acid or basic solutes can be separated with no loss in resolution.As the concentration of solute increases, the separation simply producesa wider plateau for each solute.

With an increasing demand for optically active compounds, thedevelopment of methods for the separation of enantiomers is beingintensively pursued. The preparation of optically active compounds hasbecome very important for the development of new biologically activesubstances containing one or several chiral centers, because many chiraldrugs display different activity and toxicity profiles with respect totheir absolute configuration.

The direct separation of enantiomers by chromatography is now widelyused and a large number of chiral columns using a solid support chiralstationary phase becomes more and more popular and diversified. Morethan one hundred chiral stationary phases are commercially availableallowing many analytical problems to be solved. However, few preparativeapplications have been reported because of the limited capacity of thestandard size columns. Large columns are very expensive.

Compared to the rapid development of optical isomer separation by liquidcolumn chromatography using chiral stationary phases, little work hasbeen reported concerning the separation of optical isomers bycountercurrent chromatography. Recently, others have successfullyseparated D,L-amino acid derivatives by centrifugal partitionchromatography. See, Oliveros, et al., J. Liq. Chromatogr. 17:2301(1994). However, this method can only be applied to microgram quantitiesof samples. Moreover, the chromatographic fractions isolated byOliveros, et al. were contaminated with substantial amounts of chiralselector and further purification was required. This may also be aproblem in other liquid-liquid chromatography techniques.

SUMMARY OF THE INVENTION

The present invention provides methods for the preparative-scaleseparation of optical isomers of a racemic pair using high-speedcountercurrent chromatography (HSCCC). In one embodiment, a chiralselector is held in a liquid stationary phase through which a mobilephase flows, the chromatographic process taking place between the twoliquid phases. The separations are carried out with a two-phase solventsystem in which the chiral selector is distributed almost exclusively inthe stationary phase while the analytes are partitioned between the twophases. The column is first filled with the stationary phase containingthe chiral selector, followed by sample injection. The mobile phase isthen eluted through the column. A racemic mixture of enantiomers isresolved according to the difference in affinities of the D- and L-forms(or (+) and (−) forms) to the chiral selector. As is a common practicein high performance liquid chromatography (HPLC), the CCC separation canbe repeated by successive sample injection without renewing the columncontents. The advantage of the present method is derived from the factthat the column contains no solid support and there is no need toimmobilize the chiral selector to the solid stationary phase whichinvolves a complicated synthetic process. The liquid stationary phasecan hold a large amount of the chiral selector compared to the solidsupport chiral stationary phase within the conventional chromatographiccolumn. The sample loading capacity and resolution of racemates dependnot only on the column volume but also on the concentration of thechiral selector in the stationary phase. Consequently, in HSCCC thechiral separation can be applied in both analytical and preparativescales using the same column only by adjusting the concentration ofchiral selector in the stationary phase.

The present invention also provides methods for the preparative-scaleseparation of optical isomers of a racemic pair using pH-zone-refiningcountercurrent chromatography.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows two HPLC chromatograms from chiral separations of (±)3,5-dinitrobenzoyl-leucine by HSCCC. In FIG. 1A, the two-phase solventsystem was composed of hexane:ethyl acetate:methanol:water (6:4:5:5). InFIG. 1B, a solvent system of hexane:ethyl acetate:methanol: 10 mM HCl(6:4:5:5) was used. For both separations, the apparatus was a multilayercoil high-speed CCC centrifuge with a semipreparative column of 1.6 mmI.D. and 325 mL capacity. N-dodecanoyl-L-proline-3,5-dimethylanilide (30mM) was added to the upper stationary phase as a chiral selector forboth separations. Samples of DNB-Leu (500 mg) were dissolved in 30 mLsolvent (15 mL of each phase). Other parameters included a flow rate of3.0 mL/min in the head to tail elution mode; revolution of 800 rpm; anda stationary phase retention of 65% of the total column capacity.

FIG. 2 shows chromatograms from the chiral separation of a set ofDNB-amino acids by HSCCC. In FIG. 2A, a sample of (±) DNB-phenylglycinewas used. FIG. 2B shows the separation achieved using a 10 mg sample of(±) DNB-phenylalanine. FIG. 2C shows the separation of 10 mg each of (±)DNB-valine and (±) DNB-leucine. The samples were each dissolved in 5 mLsolvent (2.5 mL of each phase) using a solvent system composed ofhexane:ethyl acetate: methanol:0.1 M HCl (6:4:5:5). Other experimentalconditions were as follows: Apparatus: Multilayer coil high-speed CCCcentrifuge with a semipreparative column of 1.6 mm ID and 325 mLcapacity; N-dodecanoyl-L-proline-3,5-dimethylanilide (20 mM) was addedin the upper stationary phase as a chiral selector; Flow rate: 3.0mL/min in the head to tail elution mode; Revolution: 800 rpm; stationaryphase retention: 65% of the total column capacity.

FIG. 3 shows six HPLC chromatograms which illustrate the effect ofvarying the amount of the chiral selector in the stationary phase forthe chiral separations of (±) DNB-leucine using HSCCC. The apparatusused was a Multilayer coil highspeed CCC centrifuge with asemipreparative column of 1.6 mm ID and 325 mL capacity. The solventsystem was composed of hexane/ethyl acetate/methanol/10 mM HCl(6:4:5:5). N-dodecanoyl-L-proline- 3,5-dimethylanilide was added to theupper stationary phase as a chiral-selector at concentrations of 10 mM(A), 30 mM (B) and 60 mM (C). Samples of (±)DNB-Leu were (A) 125 mg and250 mg, (B & C) 500 mg and 1000 mg dissolved in 10-45 mL of solvent(equal volumes of each phase). Other parameters were as described underFIG. 1.

FIG. 4 shows the HPLC chromatograms from the separation of (±)DNB-Leucine using pH-zone-refining CCC. The experimental conditions forpH-zone-refining CCC included use of a Multilayer coil high-speed CCCcentrifuge with a semipreparative column of 1.6 mm ID and 325 mLcapacity; a solvent system composed of methyl t-butyl ether/H₂O with thestationary phase being the upper organic phase to which 20 mMtrifluoroacetic acid and 40 mM chiral selector were added. The mobilephase was the lower aqueous phase to which aqueous ammonia was added at20 mM. A sample size of (±) DNB-Leucine of 2 g was used. See caption forFIG. 2 for the experimental conditions of analytical CCC. Note that theanalysis of the fraction from the mixing zone (middle chromatogram)shows three peaks corresponding to (−) DNB-Leu, impurity, and (+)DNB-Leu from left to right.

DETAILED DESCRIPTION OF THE INVENTION

The following abbreviations are used herein: DNB, dinitrobenzoyl; HPLC,high performance liquid chromatography; HSCCC, high-speed countercurrentchromatography; I.D., inside diameter; Leu, leucine; Phe, phenylalanine;Phg, phenylglycine; PTFE, polytetrafluoroethylene; TLC, thin layerchromatography; and Val, valine.

As used herein, the term “racemic” is used to indicate that twoenantiomeric forms of a compound are present together in either solid orliquid form. A racemic mixture is optically inactive, but is capable ofbeing separated into dextro- and levorotatory forms. The racemic form ofa compound is often denoted as (±), while the optically activeenantiomeric forms are denoted separately as (+) and (−). Alternatively,a racemic mixture can be referred to as a dl-mixture or DL, with theseparated forms noted as D- and L-.

As used herein, the term “chiral selector” is used to refer to acompound which is chiral (i.e., rotates polarized light) and whichinteracts more favorably with one member of an enantiomeric pair thanwith the other member. A chiral selector will typically be one opticalisomer of an α-amino acid which is suitably derivatized to provide acompound which partitions more favorably into the stationary liquidphase which is used in a separation. Examples of derivatization includeacylation of the α-amino group with a long chain fatty acid (i.e.,dodecanoic acid) and/or esterification or amidation of the carboxylicacid residue. In instances in which the chiral selector is partitionedpreferably into an organic liquid phase, the carboxylic acid residuewill be amidated with a hydrophobic group such as 3,5-dimethylaniline.Other suitable hydrophobic amides, esters and acylated amines can alsobe prepared. Examples of these are known to those of skill in the artand can be found in, for example, Greene, et al., Protecting Groups inOrganic Synthesis, Second Edition, Wiley-Interscience, New York, N.Y.,(1991), Chapters 5 and 7.

As used herein, the term “separating” means to increase the amount ofone component relative to the amounts of other components in a samplemixture. The mixture produced upon “separating” one component will besubstantially free from the other components in the sample mixture, butmay contain added quantities of solvents.

As used herein, the phrase “immiscible liquid phases” refers to liquidswhich may be partially miscible, but which separate into two phaseshaving a liquid interface on standing. Typically, the two phases willcomprise an organic phase and an aqueous phase. Suitable organicsolvents include diethyl ether, hexane, ethyl acetate, methanol, methylt-butyl ether, and acetonitrile.

As used herein, the term “identifying” means determining byspectroscopic means such as UV detection, refractive index detection,mass spectroscopy, and IR detection whether the desired compound ispresent in a particular sample or eluted fraction. Compounds may also be“identified” by a comparison of their elution times using HPLC.

The method of the present invention utilizes a countercurrentchromatographic centrifuge which may be any of the centrifuges generallyused in other modes of countercurrent chromatography. A variety of thesecentrifuges have been described by Ito, Y., in “Principle andInstrumentation of Countercurrent Chromatography,” in CountercurrentChromatography: Theory and Practice Mandava, N. B., and Ito, Y., eds.,pp. 79-442 (Marcel Dekker, New York, 1988) and by Conway, W. D., inCountercurrent Chromatography: Apparatus, Theory and Applications (VCH,New York, 1990).

Countercurrent chromatography utilizes the hydrodynamic behavior of twoimmiscible solvent phases mixing in a column to effect the separation ofa solute from other components in a sample.

For any of the methods of the present invention, any mixture of solventswhich forms two phases on standing may be used. The phases may each beindependently composed of organic solutions or aqueous solutions. In apreferred embodiment, one phase is composed of one or more organicsolvents and the other phase is substantially aqueous. Whenchromatography is conducted with the aid of a centrifuge, preferredsolvents are those which form two phases having a difference in densityof at least 0.05 g/mL. The phases may be equilibrated relative to oneanother either prior to or during chromatography. In those methods usingan acidified or basified liquid phase(s), the two phases may beequilibrated prior to acidifying or basifying the separate phases. Whena basic aqueous phase is used as a mobile phase for separation of acidicsolutes, the phases may be equilibrated after the aqueous phase is madebasic. Similarly, when an acidic aqueous phase is used as a mobile phasefor the separation of basic solutes, the phases may be equilibratedafter the aqueous phase is made acidic. In a preferred embodiment, thephases are equilibrated in their neutral form by shaking them togetherand then allowing them to separate prior to charging the column with thestationary phase. When the phases are equilibrated in their neutralform, the stationary phase may be acidified (for separation of acidicsolutes) or basified (for separation of basic solutes) prior to chargingthe chromatography column.

The selection of the solvent system for chiral separations using HSCCCis primarily based on the partition coefficients (K) between the twophases of both the chiral selector and the analyte of interest.Preferably, the chiral selector will be distributed mainly into thestationary phase, whereas the analytes should have K values of about 0.3to 1.0 so that they are rather evenly distributed between the twophases. In a preferred embodiment, the solvent system is composed ofhexane, ethyl acetate, methanol and water. More preferably, the solventsystem is composed of hexane, ethyl acetate, methanol and 10 mM HCl.

The selection of the solvent system for chiral separations usingpH-zone-refining CCC will be as described in U.S. Pat. No. 5,332,504.Briefly, the degree of acidity and basicity of the two phases is notcritical. In most applications, best results will be achieved by usingan acidic phase with a pH below about 4 and preferably below about 3.Similarly, the basic phase will in most cases have a pH above about 8and preferably above about 9. The use of a more basic mobile phase willresult in shorter elution times for acidic compounds. Similarly, a moreacidic mobile phase will reduce the elution times of basic samples.

The motions which are applied to a CCC column are best described ascorresponding to a solar system. In particular, a coiled column mayundergo rotation about one or more axes. Solar coaxial motion is foundwhen the coiled column is rotated about the axis of the coil. When thecoil is mounted with its axis parallel and offset from a second axis,and the column is rotated only about the second axis, the rotation istermed solar satellite or solar eccentric motion. Planetary motion isprovided when rotation occurs about two axes. When a coiled column isrotated about its own axis and also rotated about a second parallelaxis, the motion is termed planetary coaxial motion. When a coiledcolumn is rotated about a first external axis parallel to the axis ofthe coiled column, and the first external axis is simultaneouslyrevolving about a second external parallel axis, the motion is termedplanetary satellite or planetary eccentric motion.

In addition to configurations having parallel axes, there are alsoconfigurations in which the column axis is inclined or skewed relativeto the external axes. Another type of planetary motion results when thetwo axes about which rotation occurs are orthogonal to one another.Methods utilizing this type of configuration are termed cross-axis CCC.

The columns employed in CCC are equally diverse. The majority arehelical, but may vary in the material of fabrication, length, width,pitch of its winding, and mounting onto a column holder. Modern columnsare typically constructed of polytetrafluoroethylene tubing which iscapable of maintaining its shape and integrity while being exposed to astrong acceleration field. The inside diameter of the tubing istypically between 0.75 and 3 mm. While a single-layer coil may involveonly a few meters of tubing, a multi-layer coil might contain more than100 m of the tubing. Columns to be used for analytical purposestypically have an inside diameter which is more narrow and a lengthwhich is longer than a column used for preparative purposes.Additionally, helical columns may be either right-handed or left-handed.The handedness of the coils are determined by the direction in which thecoils are wound onto a spool-shaped column holder. The helical columnmay be either a single layer or multilayer coil. For another columnshape, the tubing may be wound onto a flexible core which is in turncoiled onto the column holder to produce a toroidal coil. Yet anothertype of column is a single layer spiral in which the tubing is wound inone layer onto a core and upon itself. The columns are further equippedwith flow tubes which provide for the introduction of sample and mobilephases using an external pump. The tubes further allow the eluate to becollected using an automated fraction collector.

The present invention can be used with any of the columns and motionsemployed for CCC. The preferred apparatus is a high-speed countercurrentchromatographic centrifuge having a multilayer-coil separation column.The preferred motion is planetary motion (either coaxial or eccentric).Particularly preferred is synchronous planetary motion in which thenumber of revolutions about each of the two axes of rotation is the samewithin a particular period of time. The synchronous planetary motionprovided by the centrifuge performs two functions. First, thesynchronous rotation of the column holder constantly unwinds the twistof the flow tubes caused by revolution. This permits continuous elutionthrough the rotating coil without the use of a conventional rotary sealdevice, which can be a potential source of leakage and contamination ofcollected fractions. Additionally, when the coiled column is coaxiallymounted about the coil holder, the planetary motion of the holderunilaterally distributes two solvent phases in the column in such a waythat one phase occupies the head side, and the other phase occupies thetail side of the coil. This head-tail relationship refers to theArchimedean screw force acting on the rotating coil, where all objectsof different density are driven from the tail portion of the coil towardthe head of the coil. This hydrodynamic phenomenon can be utilized forperforming CCC in two ways. The coil can be entirely filled with a firstliquid phase and eluted with the second liquid phase from the tailtoward the head. Alternatively, the coil can be filled with the secondliquid phase followed by elution with the first liquid phase from thehead toward the tail. In either case the hydrodynamic phenomenonfacilitates rapid movement of the mobile phase through the stationaryphase, yielding extremely high retention of the stationary phase in thecoil.

In one group of embodiments, the present inventive method is used toseparate the enantiomers of a racemic compound mixture from each otherin a sample mixture using HSCCC. Two immiscible solvent phases areequilibrated relative to one another to yield a two-phase mixture. Acountercurrent chromatographic centrifuge column is then charged with afirst liquid phase of the mixture. The first liquid phase is chargedwith a chiral selector, and the sample mixture containing the racemiccompound to be separated is introduced into the column. Alternatively,the chiral selector is added to the stationary phase (first liquidphase) prior to its introduction to the column. The centrifuge isstarted and the second liquid phase, or mobile phase, is passed throughthe column. Fractions containing the various components of the mixtureare eluted, collected and identified.

The liquid phases are each independently an organic phase or an aqueousphase. In a preferred embodiment, the first liquid phase is an organicphase and the second liquid phase is an aqueous phase. In a furtherpreferred embodiment, the first liquid phase is an organic phase and thesecond liquid phase is an acidic aqueous phase.

As noted above, the chiral selector is typically selected so that it isretained primarily in the first liquid phase (the stationary phase). Inthose applications in which the stationary phase is an organic phase,the chiral selector will be a hydrophobic compound, such as aderivatized amino acid, preferablyN-dodecanoyl-L-proline-3,5-dimethylanilide. Alternatively, inseparations in which the stationary phase is aqueous, the chiralselector will be more hydrophilic. The concentration of chiral selectorwhich is used is typically from about 0.1 to about 200 mM, preferablyfrom about 1.0 to about 100 mM, and more preferably from about 10 toabout 50 mM.

The amount of a racemic compound which is to be separated into its twoenantiomers will typically be from about 1 mg to about 1 kg, preferablyfrom about 0.05 grams to about 50 grams, more preferably from about 0.5to about 10 grams, and still more preferably from about 1.0 to about 5.0grams.

In another group of embodiments, the present inventive method is used toseparate the enantiomers of a racemic acidic compound mixture from eachother in a sample mixture using pH-zone-refining CCC. In this method, achiral selector and an acid are added to a first liquid phase of twopre-equilibrated immiscible liquid phases. A countercurrentchromatographic centrifuge column is then charged with the first liquidphase. Base is then added to the second liquid phase of the twopre-equilibrated immiscible liquid phases to form a basic mobile phase.The racemic acidic compound mixture is introduced into the injectionport of the countercurrent chromatographic centrifuge column and thebasic mobile phase is passed through the countercurrent chromatographiccentrifuge column to elute, in a separated form, the (+) enantiomer andthe (−) enantiomer of the racemic acidic compound.

The amount of racemic compound which can be separated into its componentenantiomers using this method is, as above, from about 1 mg to 1 kgquantities. Preferably, the amount to be separated is from about 0.05 to50 grams, and more preferably from about 1.0 to about 10 grams. In astill further preferred embodiment, the method can be used forseparating a quantity of an acidic compound in a suspension.

As above, the liquid phases are each independently an organic phase oran aqueous phase. In a preferred embodiment, the first liquid phase isan organic phase and the second liquid phase is an aqueous phase. In afurther preferred embodiment, the first liquid phase is made acidic withan organic acid which is either acetic acid, trifluoroacetic acid,propionic acid or butanoic acid. In a still further preferredembodiment, the first liquid phase is made acidic with trifluoroaceticacid. In another preferred embodiment, the second liquid phase is madebasic with either ammonia or NaOH, more preferably ammonia.

The chiral selector used in this group of embodiments is also selectedso that it is retained primarily in the first liquid phase (thestationary phase). In those applications in which the stationary phaseis an organic phase, the chiral selector will be a hydrophobic compound,such as a derivatized amino acid, preferablyN-dodecanoyl-L-proline-3,5-dimethylanilide.

In yet another group of embodiments, the present inventive method isused for separating a quantity of the (+) and (−) enantiomers of aracemic basic compound mixture from each other using pH-zone-refiningCCC. In these embodiments, as above, two immiscible liquid phases areequilibrated relative to one another, then separated. A countercurrentchromatographic centrifuge column is charged with a first liquid phasewhich is made basic either prior to or following its introduction intothe column. The first liquid phase is also charged with a chiralselector. The mixture containing a quantity of a racemic basic compoundto be separated is then introduced into the column. The centrifuge isstarted and the second liquid phase, which has previously been madeacidic, is passed through the column. Fractions containing the variouscomponents of the mixture are eluted, collected and identified.

As above, the liquid phases are each independently an organic phase oran aqueous phase. In a preferred embodiment, the first liquid phase isan organic phase and the second liquid phase is an aqueous phase. Theliquid phases will typically be acidified or basified as required usingthe acids and bases described above. Additionally, the chiral selectorused will be as described above for the racemic acidic compound mixture.In another preferred embodiment, pH-zone-refining countercurrentchromatography can be conducted on a preparative scale using 1 mg to 1kg of the mixture containing the racemic basic compound which is to beseparated. In a still further preferred embodiment, the method can beused for separating a quantity of a racemic basic compound in asuspension.

The following examples are offered by way of illustration and are notmeant to limit the scope of the invention.

EXAMPLES Apparatus

A commercial model (Ito multilayer coil separator/extractor, P.C. Inc.,Potomac, Md., USA) of the high-speed CCC centrifuge was used throughoutthe present studies. The detailed design of the apparatus was givenelsewhere (U.S. Pat. No. 4,430,216). The apparatus holds a multilayercoil separation column and a counterweight symmetrically at a distanceof 10 cm from the central axis of the centrifuge. The column holder isequipped with a plastic planetary gear which is engaged to an identicalstationary sun gear mounted around the central axis of the apparatus.This gear arrangement produces the desired planetary motion to thecolumn holder, i.e. rotation about its own axis and revolution aroundthe centrifuge axis in the same direction at the same rate. Thisplanetary motion also prevents the flow tubes from twisting duringrevolution, thus permitting the elution of the mobile phase through thecolumn without the use of rotary seals.

The separation column consists of a single piece of 1.6 mm ID, 160 mlong PTFE (polytetrafluoroethylene) tubing (Zeus Industrial products,Raritan, N.J., USA) wound around the column holder hub with 16 layersand 325 mL capacity. Each terminal of the column was connected to a flowtube (0.85 mm ID PTFE) (Zeus Industrial Products) by the aid of a set oftube connectors (Upchurch Scientific Co., Oak Harbor, Wash., USA) whichwere rigidly mounted on the holder flange. A narrow-bore PTFE tube (0.3mm ID ×5 m)(Zeus Industrial Products) was placed at the outlet of thecolumn to stabilize the effluent flow, thus facilitating the recordingthe elution curves.

The speed of the apparatus was regulated with a speed controller (BodineElectric Company, North Chicago, Ill., USA).

Reagents

Methanol was glass-distilled chromatographic grade (Baxter HealthcareCorporation, Muskegon, Mich., USA). HPLC grade of hexane and ethylacetate, and reagent grade of sodium hydroxide and hydrochloric acidwere purchased from Fisher Scientific Company (Fair Lawn, N.J., USA).Dinitrobenzoyl(DNB)-leucine, DNB-phenylglycine and 3,5-DNB chloride wereobtained from Aldrich Chemical Co., (Milwaukee, Wis., USA). DL-proline,DL-valine, DL-phenylalanine, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydrofuran(EEDQ), dodecanoyl chloride, 3,5-dimethylaniline were purchased fromSpectrum Chemical Mfg. Corp. (New Brunswick, N.J., USA).

Synthesis of Chiral Selector

N-dodecanoyl-L-proline: L-proline (11.6 g, 0.1 mol) was dissolved in 150mL of 1 M NaOH solution and cooled in an ice both. To this solutiondodecanoyl chloride (24.1 g or O.11 mol) and 200 mL of 1 M NaOH solutionwere added simultaneously over a period of 20 min. The solution wasstirred at room temperature for 45 min and acidified with concentratedHCl (pH 2-3). The solution was extracted with diethyl ether and theorganic phase was washed with 12% NaCl solution. The organic phase wasthen dried over sodium sulfate and evaporated to provide 30 g ofN-dodecanoyl-L-proline as an oil (100% yield) which was carried onwithout purification.

N-dodecanoyl-L-proline-3,5-dimethylanilide: To 400 mL of a THF solutioncontaining 13.0 g (0.11 mol) of freshly distilled 3,5-dimethylanilineand 30 g (0.1 mol) of N-dodecanoyl-L-proline, was added 200 mL of THFcontaining 25.2 g (0.1 mol) of EEDQ at room temperature. The mixture waskept at room temperature for 24 hr and the solvent was evaporated. Theresidue was redissolved in dichloromethane and washed with 1%orthophosphoric acid, 0.2 M NaOH and then distilled water. The resultingsolution was dried over sodium sulfate, filtered and evaporated.Recrystallization of the residue from ethanol-water gave 35.0 g (87%yield) of white solid.

Another chiral selector which is useful for the separation of naproxenisomers isN-(2′,6′-dimethylpiperidine)-6-methoxy-α-methyl-2-naphthanleneethanamide. Synthesis of this chiral selector was carried out asfollows:

(S)-naproxen, (S-(+)-methoxy-α-methyl-2-naphthalene acetic acid), (2.30g, 1 mmol) was dissolved in 50 mL of methylene chloride and 2 mL ofoxalyl chloride was added. After 3 hours, the solution was evaporated todryness and the remaining crystalline solid was dried under vacuum for 3hours. The solid was dissolved in 100 mL of dry methylene chloride andcooled in an ice-water bath. 2,6-dimethylpiperidine (13.5 mL, 100 mmol)was added slowly with stirring, and the mixture was kept at roomtemperature for 2 hours. The resulting mixture was washed successivelywith 1M HCl (100 mL), 1% NaOH (100 mL) and water, then dried overmagnesium sulfate. Removal of solvent under reduced pressure provided3.2 g (98% yield) ofN-(2′,6′-dimethylpiperidine)-6-methoxy-α-methyl-2-naphthanleneethanamide.

Synthesis of Analytes

(±) N-(3,5-dinitrobenzoyl)amino acids: An appropriate amount of racemicamino acid (23 mmol) was dissolved in 50 mL of 1M NaOH solution andcooled in an ice-bath. To this solution 3,5 -dinitrobenzoyl chloride(5.3 g, 23 mmol) and 50 mL of 1M NaOH solution were added simultaneouslyover a period of 20 min. The resulting solution was stirred at roomtemperature for 90 min and acidified with concentrated HCl (pH 2-3). Theresulting solid was collected by filtration and washed with water.Recrystallization of the solid from ethanol-water gave the correspondingDNB-amino acid.

Preparation of Solvent Phases and Sample Solutions

The solvent pair was prepared as follows: hexane, ethyl acetate,methanol and distilled water (or dilute HCl solution) were thoroughlyequilibrated in a separatory funnel at room temperature and the twophases were separated. The chiral selector(N-dodecanoyl-L-proline-3,5-dimethylanilide, 10-60 mM) was added to theupper organic phase which was then used as the stationary phase. Thelower aqueous phase was used as the mobile phase.

Sample solutions were prepared by dissolving a set of DNB-amino acids in10-50 mL of solvent typically consisting of about equal volumes of upperorganic and lower aqueous phases. The solution was sonicated for severalminutes before injecting into the column.

Separation Procedure

In each separation, about 150 mL of the organic stationary phase free ofchiral selector was first pumped into the column followed by 200 mL ofthe same organic phase but containing the chiral selector at a desiredconcentration. The sample solution was then injected through the sampleport and the aqueous mobile phase was eluted through the column in thehead to tail elution mode at a flow rate of 3.0 mL/min (Rainin MeteringPump: Rainin Instruments Co. (Emeryville, Calif., USA)) while theapparatus rotated at 800 rpm. The effluent from the column wascontinuously monitored by absorbance at 254 nm (Uvicord S, LKBInstruments, Bromma/Stockholm, Sweden) and collected at 3.0 mL/tube(Ultrorac Fraction Collector, LKB Instruments). After all peaks wereeluted, the centrifuge run was terminated, and the column contents werecollected into a graduated cylinder by connecting the inlet of thecolumn to a nitrogen line at 80 psi. The retention of the stationaryphase relative to the total column capacity was computed from the volumeof the stationary phase collected from the column (65-80%).

Analysis of CCC Fractions

CCC fractions were analyzed by TLC on Kieselgel 60 F254 withheptane:ethyl acetate (1:1) as eluent. The enantiomer purity of theDNB-amino acids was determined by optical rotation and circulardichroism.

Example 1

This example illustrates the separation of (±)-DNB-Leucine using HSCCC.

Complete separation of 500 mg of N-(3,5-dinitrobenzoyl)-leucine wasachieved by HSCCC using a multilayer coil planet centrifuge equippedwith a 325 mL capacity column (FIG. 1). The separation was carried outwith a two-phase solvent system composed of hexane, ethyl acetate,methanol and water (A) or 10 mM HCl (B) (6:4:5:5, by volume) whereN-dodecanoyl-L-proline-3,5-dimethylanilide was added in the upperstationary phase as a chiral selector. The mechanism of the separationis similar to that recently described by Pirkle et al, J. Chromatogr.,641:11 (1993) in which the chiral selector was chemically bound to thesurface of silica gel. The chromatogram (A) obtained by the neutralmobile phase shows skewed peaks which lead to incomplete peakresolution. The peak resolution was substantially improved by using anacidic mobile phase (B) because at the lower pH all analyte moleculesare protonated and distributed by a uniform K value according to thelinear isotherm.

In HSCCC the selection of the solvent system for chiral separation ismainly based on the partition coefficients (K) of both the chiralselector and analytes. The partition coefficient (K) is defined asC_(s)/C_(m), where C_(s) is the concentration in the stationary phaseand C_(m) is the concentration in the mobile phase. The chiral selectorshould have a large K value so that it is unilaterally distributed tothe organic stationary phase whereas the analytes should have K valuesof near unity to be rather evenly partitioned between the two phases. Inthe present example this requirement was fulfilled by binding adodecanoyl group to the L-proline-3,5-dimethylanilide to increase thehydrophobicity of the selector molecule. In the solvent system composedof hexane, ethyl acetate, methanol and 10 mM HCl (6:4:5:5) used in theabove separation, the partition coefficient of the chiral selector is aslarge as 45.1 while DNB-Leucine has a partition coefficient of 1.3.

Contamination of the chiral selector in the eluted fractions was furtherprevented by filling the column only partially with the stationary phasecontaining the chiral selector while leaving the end of the column spacewith the same phase free of the chiral selector. In this way a smallamount of the chiral selector carried by the flowing mobile phase isabsorbed into the stationary phase near the end of the column.

Example 2

This example illustrates the separations which are achieved usingracemic mixtures of DNB-phenylglycine, -phenylalanine, -leucine, and-valine and correlates the amount of separation with the nature of theside chain and the partition coefficient, K. The partition coefficientis calculated as the concentration of the analyte in the stationaryphase (C_(a)) divided by the concentration of the analyte in the mobilephase (C_(m)).

The enantiomers of the homologous series of DNB-amino acids wereseparated with the same system on an analytical scale (10 mg) in orderto investigate the mechanism of enantioselectivity of the chiralselector in HSCCC (FIG. 2). The separations were performed in thesolvent system composed of hexane/ethyl acetate/methanol/10mM HCl(6:4:5:5) where N-dodecanoyl-L-proline-3,5-dimethylanilide were againused as a chiral selector in the upper stationary phase. Chromatograms Aand B were obtained by injecting the racemic mixture of DNB-Phg andDNB-Phe, respectively, and chromatogram C by simultaneous injection ofthe racemic mixtures of DNB-Leu and -Val. The separation was repeated bysuccessive sample injection without renewing the column contents. Theresults indicate the enantioselectivity of DNB-amino acids is related tothe side chain (R) of the molecule (Table 1). The chromatographicseparation factor, which is determined from the ratio of retention timebetween two enantiomers (here the retention time is the time required toelute the analytes after the solvent front has emerged) increases withthe increasing size of R, while it also relates to hydrophobicity of thesolvent system and the concentration of chiral selector in stationaryphase.

TABLE 1 Separation Factors of Enantiomers of DNB-Amino Acids Sample R K= C_(s)/C_(m)* Separation factor DNB-Leucine —CH₂CH(CH₃)₂ 1.24 1.76DNB-Valine —CH(CH₃)₂ 0.78 1.38 DNB-Phenylalanine —CH₂C₆H₅ 1.08 1.71DNB-Phenylglycine —C₆H₅ 0.93 1.25 *K indicates the partition coefficientof enantiomers before the chiral selector is added to the solventsystem.

Example 3

This example illustrates the effect of the chiral selector concentrationon the separation of (±)-DNB-Leucine.

The sample loading capacity of the present method was investigated withthe separation of DNB-leu by varying the concentration of the chiralselector in the stationary phase. As shown in FIG. 3, concentrations ofthe chiral selector at 10, 30 and 60 mM in the stationary phase wereexamined in the same solvent system composed of hexane/ethylacetate/methanol/10 mM HCl (6:4:5:5). The result indicated that thesample loading capacity is largely determined by the concentration ofthe chiral selector in the stationary phase: The higher theconcentration of the chiral selector, the better peak resolution isobtained.

These results clearly demonstrate an important advantage of HSCCC in theseparation of enantiomers. The same column can be applied to bothanalytical and preparative scale separations by choosing differentconcentrations of chiral selector in the stationary phase.

Example 4

This example illustrates the separation of (±)-DNB-Leucine usingpH-zone-refining countercurrent chromatography.

pH-Zone-refining CCC is a powerful preparative method comparable todisplacement chromatography. The method yields a succession of highlyconcentrated rectangular solute peaks with minimum overlap whereimpurities are concentrated at the peak boundaries. See, U.S. Pat. No.5,332,504, incorporated herein by reference.

The separation of chiral compounds using pH-zone-refining CCC wasperformed with a binary two-phase solvent system composed of methylt-butyl ether and water. After the two phases were separated, properamounts of a retainer acid (trifluoroacetic acid, 20 mM) and the chiralselector (N-dodecanoyl-L-proline-3,5-dimethylanilide, 40 mM) were addedto the organic stationary phase and an eluent base (ammonia) was addedto the aqueous mobile phase. The column was first completely filled withthe organic stationary phase followed by injection of the samplesolution containing 2 g of (±) DNB-leu. The mobile phase was then elutedthrough the column while the apparatus was rotated at 800 rpm. Theeffluent from the outlet of the column was continuously monitored with aUV monitor (Uvicord S) at 206 nm and fractionated into test tubes (3mL/tube). FIG. 4 shows a typical chromatogram obtained by the presentmethod. The analyte was eluted in a single rectangular peak which isevenly divided by two pH zones. The separation was completed in about 3hours. Peak fractions were analyzed by analytical scale CCC as describedearlier. The results showed that the first part was almost entirelycomposed of (−) DNB-leu and the second part from (+) DNP-leu which themiddle portion of the peak contained both isomers and an impurity asshown in the upper portion of the diagram (FIG. 4). The mixing zone ofthe two peaks is estimated less than 5% of each peak.

Compared with the standard CCC technique described earlier, thepH-zone-refining CCC technique enables the separation of larger amountsof the sample in a shorter period of time. In addition, the method usesa relatively polar solvent system which can hold a chiral selector for amuch longer period of time, thus reducing the risk of its contaminatingthe effluent fractions.

The overall results of our studies indicate that the present method canbe utilized for chiral separation of gram quantities of DNB-amino acids.The method may be extended to the separation of various chiral compoundsusing appropriate chiral selectors.

The above description is illustrative and not restrictive. Manyvariations of the invention will become apparent to those of skill inthe art upon review of this disclosure. Merely by way of example avariety of analytes, chiral selectors, liquid phases and mixtures, andother materials may be used without departing from the scope of theinvention. The scope of the invention should, therefore, be determinednot with reference to the above description, but instead should bedetermined with reference to the appended claims along with their fullscope of equivalents.

What is claimed is:
 1. A method for separating a quantity of the (+) and(−) enantiomers of a racemic acidic compound mixture from each otherusing pH-zone-refining countercurrent chromatography, comprising: (a)adding a chiral selector and acid to a first liquid phase of twopre-equilibrated immiscible liquid phases and charging a countercurrentchromatographic centrifuge column with said first liquid phase, therebyproducing a countercurrent chromatographic centrifuge column chargedwith said chiral selector and said thus acidified first liquid phase;(b) adding base to a second liquid phase of said two pre-equilibratedimmiscible liquid phases to form a basic mobile phase; (c) introducingsaid racemic acidic compound mixture into said countercurrentchromatographic centrifuge column thus charged with said chiral selectorand said acidified first liquid phase; and (d) passing said basic mobilephase through said countercurrent chromatographic centrifuge column thuscharged with said mixture, said chiral selector and said acidified firstliquid phase, to elute said (+) enantiomer and said (−) enantiomer ofsaid racemic acidic compound from said countercurrent chromatographiccentrifuge column, wherein said quantity is from 1 mg to 1 kg.
 2. Amethod in accordance with claim 1 wherein said first liquid phase is anorganic phase and said second liquid phase is an aqueous phase.
 3. Amethod in accordance with claim 1 wherein said acid is selected from thegroup consisting of trifluoroacetic acid, acetic acid, propionic acidand butanoic acid.
 4. A method in accordance with claim 1 wherein saidacid is trifluoroacetic acid and said base is ammonia.
 5. A method inaccordance with claim 1 wherein said acid is trifluoroacetic acid, saidbase is ammonia and said chiral selector isN-dodecanoyl-L-proline-3,5-dimethylanilide.
 6. A method in accordancewith claim 1 wherein said racemic acidic compound mixture is a mixtureconsisting essentially of a (±) N-protected amino acid.
 7. A method inaccordance with claim 1 wherein said racemic acidic compound mixture isa mixture consisting essentially of a (±) N-(3,5-dinitrobenzoyl)aminoacid.
 8. A method in accordance with claim 1 wherein said quantity isfrom 1.0 to 50 g.
 9. A method in accordance with claim 1 wherein saidcolumn is a helical column.